CN103695464A - Micro RNA expression vector and application - Google Patents

Abstract

The invention discloses a micro RNA expression vector and application. The invention clones a miR-596 expressed gene fragment, and constructs a U6-miR-596 expression vector. Experiments prove that the product can inhibit propagation of lung cancer tumor cells, and has killing effect for propagation of lung cancer tumor cells, thereby inducing apoptosis of lung cancer cell line, and providing a new thinking of gene treatment of tumor.

Description

A kind of micro-RNA expression carrier and application

Technical field

The present invention relates to molecular biology and gene therapeutics field, in particular a kind of micro-RNA expression carrier and the application in suppressing lung cancer tumor cell proliferation and shifting thereof.

Background technology

MicroRNAs (miRNAs) is generally 21-23 Nucleotide, is the important small molecules non-coding RNA of a class.MiRNAs expresses has vital role, is controlling growth, differentiation and the apoptosis of cell, occurs closely related with tumour.MiRNAs normally transcribes generation by rna plymerase ii, and initial product is the large precursor molecule that is called as pri-miRNA.Pri-miRNAs is processed by Drosha (RNase III) and double-strand RNA binding protein Pasha the pre-miRNA forming into about 70m in nucleus, and this molecule is the stem ring texture of a base incomplete pairing.RAN-GTP and Exportin5 can be transported to pre-miRNA molecule in tenuigenin.This stem ring texture is double-stranded into about the ripe miRNA of 22 length of nucleotides by Dicer (another kind of RNaseIII) processing subsequently.Next the double-stranded miRNA of this maturation can be incorporated into very soon in miRISC complex body and (in complex body, contain Argonaute albumen).Ripe miRNA is attached to the mRNA site with its sequence complementation, by two kinds of expression that depend on sequence complementary mechanisms negative regulate target gene.It is to be determined by the mispairing degree between miRNA and its object mRNA that its inhibition translation or degraded play a role, and the object mRNA that matching degree is high will be degraded.

MiRNAs can play tumor suppressor gene effect, and miR-15a/miR-16 can negative regulation BCL2, therefore, the disappearance of these two miRNAs or downward, the rising that has caused BCL2 to express, can promote the generation of leukemia, lymphoma and prostate cancer.The expression level of let-7c family is significantly lowered in the kinds of tumor cells such as lung cancer, ovarian cancer, mammary cancer, and in lung cancer and mammary cancer, the expression level of let-7 and patient's survival time are proportionate.MiR-99 also can be used as and presses down the propagation that cancer miRNA suppresses tumour, causes the apoptosis of tumour cell.But relevant miR-596 research report is less, and function also needs further research.The generation of the regulatory factor of miRNA after as a kind of transcribing and tumour is closely related, and clone presses down cancer miRNA gene and aspect the gene therapy of tumour, has important value.

The method of the tumours such as existing treatment lung cancer, cancer of the stomach and cervical cancer mainly contains medicine chemotherapy, radiotherapy and operative treatment, but above-mentioned method defectiveness all, and especially for inoperable patient, chemotherapy does not reach satisfied result for the treatment of.

Therefore, there is defect in prior art, needs to improve.

Summary of the invention

Technical problem to be solved by this invention is the defect existing in treatment tumour for prior art, and a kind of micro-RNA expression carrier and the application in suppressing lung cancer tumour cell thereof are provided.

Technical scheme of the present invention is as follows:

A micro RNA expression vector; characterized in that the micro-RNA expression vector withATTACGCCAGCTTGCATGCCTGCAGGTCGACGATTCGGGACTTACAGGGAACAGCACAGCCTGGGGGCCATCGCAGATGCCCTCCAGGAAAGCAGGATGCTCTGCAGATAGGACGGCTGTGGCCGCTGCCCCAGGAAACAGCCCAGGACGCTGCCTGAGCTCAGAGGGTTCCGGACACAGACCGCGCTCCCCGCTTTGCAAACCACACCCAGTCCATTTCCTAGTGTGAGTTTGTCTCTACTCATCGGTTTGTGTCTTTATAAGAGTTCTAAAGTAAAACAAAAACACACAAGGACAGTGACCTAGACAGCACCCAGGGATGGGTCGGGTCGGCCTCCGAAGGGCAGCAGCTGCCATGCGGGAGGCAGGTTCCCGAGGAGCCGGGCAGGCTTCGGAGAGGCCGTGCTCGCTTTCCCGACTGTGGCGACGGATCCACCTGCTATGCGTTCCTCAGAACTCACTGAACCGCCCACGTTAATGACGCGCAGTTCGCCGTGTGCCAATGACGCCTCAATCAAGCTGCTAAATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACGene fragment。

The application of described micro-RNA expression carrier in suppressing lung cancer tumor cell proliferation and shifting.

The present invention has cloned miR-596 expressing gene fragment, and build U6-miR-596 expression vector, experimental results show that carrier can suppress the propagation of lung cancer tumour cell, propagation to lung cancer tumour cell has lethal effect, cause the apoptosis of lung cancer cell line, for the gene therapy of tumour provides new thinking.

Accompanying drawing explanation

Fig. 1 is U6-miR-596 expression vector establishment schematic diagram;

Fig. 2 is that quantitative PCR detection miR-596 expresses.

Fig. 3 is that MTT analyzes the growth-inhibiting situation to cell.

Fig. 4 is apoptotic detection after A549 cell transfecting carrier.

Fig. 5 is microscopic examination cell migration result.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

In the present invention, the technology of using, comprises gene sequencing, pcr amplification and detection, cell transfecting, vector construction equimolecular biology techniques, and cell cultures, detection technique etc., unless stated otherwise, be the known routine techniques of those skilled in the art; The plant and instrument using, reagent, plasmid and carrier, clone etc., only this specification sheets is dated especially, is that the research of general this area and technician can obtain by public approach.

The structure of embodiment 1U6-miR-596 expression vector

The primer sequence of design clone miR-596 expression vector, by pcr amplification miR-596 gene, use instrument is Eppendorf mastercycler gradiemPCR instrument.

Masterplate is normal people's genomic dna, and extracting method is as follows: gather 1ml peripheral blood, be placed in 1.5ml centrifuge tube (inside having appropriate anticoagulant heparin agent), 3000rpm abandons supernatant for centrifugal 5 minutes.Add 800 μ l physiological saline, put upside down and mix, centrifugal 5 minutes of 1000rpm, abandons supernatant, repeats 3 times.Add the haemolysis reagent (splitting erythrocyte is used) that is equivalent to 5.5 times of volumes of hematocrit cell ,-20 ℃ of static 10min after fully mixing, put into subsequently 4 ℃ and place 30min, and now solution becomes transparence reddish-brown.By the centrifugal 5min of this solution 1000rpm, abandon supernatant.Add 400 μ l STE damping fluid suspension white corpuscles, make it dispersed.Add Proteinase K (final concentration is 100 μ tg/ml) and 10%SDS, 37 ℃ of digestion are spent the night simultaneously.Add the saturated phenol of equal-volume, fully mix, at 4 ℃ 13, the centrifugal 12min of 000rpm; Draw supernatant to another pipe.Add equal-volume phenol/chloroform, fully mix, at 4 ℃ 13, the centrifugal 8min of 000rpm; Draw supernatant to another pipe.Add equal-volume chloroform/primary isoamyl alcohol (V:V=24:1), fully mix, at 4 ℃ 13, the centrifugal 5min of 000rpm.Draw supernatant to another pipe, add the 3M NaAc of 1/10 volume and the precooling dehydrated alcohol of 2 times of volumes ,-20 ℃ of standing 30min after mixing, 4 ℃, the centrifugal 5min of 13,000rpm, abandons supernatant.Add 1ml80% ethanol, put upside down and mix, 4 ℃ 13, the centrifugal 5min of 000rpm, abandons supernatant, and room temperature is dried, and adds appropriate 100 μ l TE to dissolve and spends the night, and spectrophotometer detects DNA concentration and purity, deposits for-20 ℃.

Amplification condition is as follows: 94 ℃ of 45s of sex change, 55 ℃ of 45s, extend 72 ℃ of 60s, totally 30 circulations.Upstream primer Sence:5 '-TAGCAGCTTGATTGAGGCG-3 '; Downstream antisense:5 '-CGGGACTTACAGGGAACAG-3 '.PCR product carries out electrophoresis, analytical results with 1.5% sepharose.Then respectively above-mentioned fragment is connected on pMD-19T (Takara company) carrier, builds miR-596, deliver to the order-checking of Shanghai Bo Shang Bioisystech Co., Ltd, result correct (SEQ ID NO:1 sequence table).Then, utilize BamHI and HindIII two enzymes (Takara company) to cut site and cut T-miR-596 and pRNAi-U6.1/Neo (Nanjing Jin Site Science and Technology Ltd.) carrier, miR-596 is connected on pRNAi-U6.1/neo carrier, obtain U6-miR-596 carrier, with same restriction enzyme site, build U6-miR-596 carrier.Vector construction flow process is shown in Fig. 1.

The impact of embodiment 2U6-miR-596 carrier on apoptosis of tumor cells

1, cell cultures and transfection

F12 (GIBICO) culture medium culturing containing 10% foetal calf serum for A549 lung carcinoma cell.Cell, purchased from cell institute of the Shanghai Chinese Academy of Sciences, is cultivated in 37 ℃ of 5%CO2.

Cell transfecting: U6-miR-596 plasmid extraction method is pressed EndoFree plasmid Maxi kit protocol (Qigen company product) operation.Adopt six well culture plates to cultivate, every hole 5 * 10 ⁵the plasmid of individual cell transfecting 1 μ g.Transfection method liposome transfection method, the ratio of plasmid and liposome is 1:3 (1 μ g:3 μ l), transfection, after 72 hours, is carried out the detection of index.

2, the detection that miR-596 expresses

MiRNA detects as follows: collecting cell, application miRNA extracts test kit (Ambion) and extracts little RNA, with carrying out RT reaction (RT primer: 5'-AACATGTACAGTCCATGGATGd (T) 30-3 ') after Polymerase A (Ambion) tailing, use respectively the corresponding miRNA of miR-596 primer amplification, and with human5S rRNA primer amplification as reference gene (upstream primer is in Table 1, and downstream primer is 5'-AACATGTACAGTCCATGGATG-3 ').Amplification condition is: 94 ℃ of 30s; 54 ℃ of annealing 30s; 72 ℃ of 30s, 29 circulations.Product is through 2% agarose gel electrophoresis analysis.Found that, after tumour cell transfection U6-miR-596 carrier, the expression amount of miR-596 obviously raises.

The expression of quantitative PCR detection miRNA: detect miRNA with the detection kit qRT-PCR miRNA Detection Kit of ambion company and express, step is with reference to specification sheets, and 5srRNA is as object of reference.Use RG3000 systems analysis.Reaction system is 20 μ l systems, comprises upstream and downstream primer, 10 * damping fluid, dNTP, high-fidelity DNA polymerase, SYBR GreenI dyestuff etc.Quantitative reaction condition is 94 ℃ of 20s, 54 ℃ of annealing 20s, 72 ℃ of 20s, each 40 circulations of increasing.When 72 ℃ of each circulation, measure fluorescence volume.Found that, after tumour cell transfection U6-miR-596 carrier, the expression amount of miR-596 obviously raises.* p<0.05: compare with control, there were significant differences for statistics.(referring to Fig. 2).

3, apoptotic detection method

The morphologic observation of 3.1 apoptotic cells

After transfection U6-miR-596 carrier 48h.The apoptosis that detects A549 cell with inverted microscope changes.

3.2MTT (3-(4,5)-dimethylthiahiazo (z-y1)-3,5-di-phenytetrazoliumromide, SIGMA company)

After transfection U6-miR-596 carrier 48h, in every porocyte, add MTT solution 10 μ l, continue to cultivate 4 hours, collecting cell, centrifugal 5 minutes of 4000rpm, abandoning supernatant, every hole adds DMSO100 μ l, after shaking up, use microplate reader to measure optical density value (570nm), detect inhibitory rate of cell growth.

3.3 apoptosis test kit Annexin V-FITC (Bao Ling Man product) detect

Attached cell is with not collecting (note: the trysinization time is difficult for long, otherwise easily cause false positive) containing the trysinization of EDTA; With PBS washed cell secondary (the centrifugal 5min of 2000rpm), collect 1～5 * 10 ⁵cell; The Binding Buffer suspension cell that adds 400 μ L; Add 5 μ L Propidium Iodide (PI, SIGMA company), mix; Room temperature, lucifuge, reaction 5-15min; In 1hour, carry out observation and the detection of following fluorescent microscope or flow cytometer.

3.4 fluidic cells detect (PI dyeing)

With flow cytometer, detect excitation wavelength Ex=488nn; Emission wavelength Em=530nm.PI red fluorescence detects by PI passage (FL2 or FL3), uses FL3.Fluorescence compensating regulation: use the normal cell of processing without apoptosis induction, carry out in contrast the position that fluorescence compensating regulation is removed spectra overlapping and set cross door.

4, the detected result of apoptosis of tumor cells

4.1miRNA cell growth form and value-added impact

After transfection U6-miR-596 carrier 48h, U6-miR-596 expression group, compared with the control, cell proliferation is subject to obvious inhibition.The propagation situation that detects cell with MTT, result shows: transfection U6-miR-596 vehicle group, Growth of Cells is subject to obvious inhibition (Fig. 3).

4.2 apoptotic cell PI dyeing and fluidic cell detected results

After transfection U6-miR-596 carrier 48h, with the apoptosis of Annexin V-FITC apoptosis kit detection cell, change, PI dyeing is used for the fracture of DNA in analysis of cells and changes.With fluorescent fiber microscopy, survey apoptotic change, in U6-miR-596 treatment group, there is obvious apoptosis in cell.And there is not obvious apoptosis in cellular control unit.U6-miR-596 treatment group is found in Flow Cytometry analysis, and obvious apoptosis (Fig. 4) appears in cell.

4.3Transwell migration experiment detects cell migration result

After cell transfecting U6-miR-596 carrier, in Transwell plate, cultivate 24h, under micro-Microscopic observation, observe the cell number of migration.Find that transfection U6-miR-596 group adjourns the cell number (Fig. 5) that the cell number of lower chamber is obviously less than blank group and negative vehicle Control group.To move to the cell dissociation of lower chamber, with Flow cytometry cell number (mean ± standard deviation), represent, get count results mean number 3 times: blank group 568 ± 42.8; Negative vehicle Control group 361 ± 32.1; Transfection U6-miR-596 group 99 ± 11.5, same visible transfection U6-miR-596 group migrating cell number is obviously less than blank group and negative vehicle Control group.

Table 1: detect the needed upstream primer of miRNAs

SEQ ID NO:1miR-596 sequence table

ATTACGCCAGCTTGCATGCCTGCAGGTCGACGATTCGGGACTTACAGGGAACAGCACAGCCTGGGGGCCATCGCAGATGCCCTCCAGGAAAGCAGGATGCTCTGCAGATAGGACGGCTGTGGCCGCTGCCCCAGGAAACAGCCCAGGACGCTGCCTGAGCTCAGAGGGTTCCGGACACAGACCGCGCTCCCCGCTTTGCAAACCACACCCAGTCCATTTCCTAGTGTGAGTTTGTCTCTACTCATCGGTTTGTGTCTTTATAAGAGTTCTAAAGTAAAACAAAAACACACAAGGACAGTGACCTAGACAGCACCCAGGGATGGGTCGGGTCGGCCTCCGAAGGGCAGCAGCTGCCATGCGGGAGGCAGGTTCCCGAGGAGCCGGGCAGGCTTCGGAGAGGCCGTGCTCGCTTTCCCGACTGTGGCGACGGATCCACCTGCTATGCGTTCCTCAGAACTCACTGAACCGCCCACGTTAATGACGCGCAGTTCGCCGTGTGCCAATGACGCCTCAATCAAGCTGCTAAATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTAC

Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (2)

1. A micro-RNA expression vector; characterized in that the micro-RNA expression vector withATTACGCCAGCTTGCATGCCTGCAGGTCGACGATTCGGGACTTACAGGGAACAGCACAGCCTGGGGGCCATCGCAGATGCCCTCCAGGAAAGCAGGATGCTCTGCAGATAGGACGGCTGTGGCCGCTGCCCCAGGAAACAGCCCAGGACGCTGCCTGAGCTCAGAGGGTTCCGGACACAGACCGCGCTCCCCGCTTTGCAAACCACACCCAGTCCATTTCCTAGTGTGAGTTTGTCTCTACTCATCGGTTTGTGTCTTTATAAGAGTTCTAAAGTAAAACAAAAACACACAAGGACAGTGACCTAGACAGCACCCAGGGATGGGTCGGGTCGGCCTCCGAAGGGCAGCAGCTGCCATGCGGGAGGCAGGTTCCCGAGGAGCCGGGCAGGCTTCGGAGAGGCCGTGCTCGCTTTCCCGACTGTGGCGACGGATCCACCTGCTATGCGTTCCTCAGAACTCACTGAACCGCCCACGTTAATGACGCGCAGTTCGCCGTGTGCCAATGACGCCTCAATCAAGCTGCTAAATCTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACGene fragment。
2. 2. the application of micro-RNA expression carrier according to claim 1 in suppressing lung cancer tumor cell proliferation and shifting.

Images